Neutrophil predominant lung inflammation is a major cause of morbidity and mortality11. Yet despite decades of investigation, accurate stratification of patients with neutrophil predominant lung injury on intensive care has been hindered by the lack of bedside point of care diagnostics that can reliably and rapidly distinguish acute neutrophilic inflammation2. The ability to perform bedside diagnostics has the potential to accurately stratify such patients for further neutrophil specific interventions. Excessive neutrophil activity degrades matrix and cellular receptors, activates profibrogenic mediators and contributes to epithelial and endothelial cell damage3,4,5. The involvement of neutrophils in several diseases such as acute lung injury, ischemia-reperfusion injury6,7, cystic fibrosis8 and chronic obstructive pulmonary disease9, makes them important targets for modulation.
In situ in vivo detection of neutrophilic inflammation in human pulmonary inflammation has been reliant upon FDG PET imaging. PET imaging10, although offering exquisite sensitivity, is cumbersome, expensive and is difficult to implement as a bedside molecular imaging modality. Conversely the advent of confocal endoscopy, such as probe based confocal laser endoscopy has revolutionised the ability to directly visualise the alveolar space in both preclinical and clinical arenas. However, as yet this modality has only been used to image autofluorescent structures within the alveolar space or using non-specific fluorescent dyes11,12.
The optical detection of activated neutrophilic activity is feasible with imaging enzymatic activity in whole animals13. These approaches require substrate specificity
with internally quenched molecular beacons. Often the dequenching may take hours and the substrate may be cleaved by non neutrophil proteases.
Dendrimers are a class of macromolecules possessing a well-defined structure and molecular composition14,15.
They are created by the stepwise attachment of monomer units in repeating unit layers, termed generations, which creates branches built upon a central core. These branches terminate in a specific chemical functional group that can be used for further dendrimer growth or modification, or attachment of specific compounds as required.
WO2003014743 describes the use of dendrimers and polybranched molecules to enhance signals in in vitro fluorescent assay systems16.
The molecules disclosed in WO2003014743 comprise cleavage sites which when treated with an appropriate chemical or enzyme lead to cleavage of selective bonds within the molecules and a subsequent change in the fluorescent properties of the molecule, most notably an increase in fluorescence. However, WO2003014743 only shows the results of in vitro data and there is no suggestion or teaching of how one might use the molecules in an in vivo setting, or indeed if this would in fact be possible.
It is an object of the present invention to obviate and/or mitigate at least one of the aforementioned disadvantages.
It is an object of the present invention to provide means of visualising cells in vivo, such as activated neutrophils within the lung of a subject, using confocal microendoscopy.
In a first aspect there is provided a dye construct which prior to cell internalisation displays substantially no detectable or only a low amount of fluorescence, but upon cell internalisation displays a detectable increase in fluorescence using confocal endoscopy for use in imaging cells in vivo:
The dye construct may be a poly-branched molecule with surface groups linked to a fluorescent dye, such as disclosed in WO/2003/014743, or a molecule as further described herein.
“Detectable increase in fluorescence” is understood to relate to fluorescence which can be detected by confocal microendoscopy techniques. If the dye constructs initially display a low, but detectable level of fluorescence, then a detectable increase can be observed following internalisation by a cell or cells. Initially the dye constructs for use in the present invention are internally quenched. That is, the dye constructs do not fluoresce or fluoresce poorly due to the fluorescent groups or groups being quenched. However, following internalisation by cells, dequenching occurs and an increase in fluorescence can be detected.
Typically fluorescence detection is understood to be related to fluorescence intensity, fluorescence lifetime and polarisation may also be detected. Typically the “low or little” amount of fluorescence” is practically not detectable using confocal microendoscopy techniques, or is sufficiently low to permit a clear identification of the “increase” in fluorescence. Typically a suitable increase is understood to be an increase by a factor of 1.2 or more.
The present inventors have observed that combining confocal endoscopic visualisation techniques with the localised administration of a dendrimer dye molecule or poly-branched molecule linked fluorescent dye, it is possible to observe in vivo, by way of fluorescence, specific cells which internalise a dye construct of the present invention or poly-branched molecule linked fluorescent dye, with a distinct increase in fluorescence. Without wishing to be bound by theory, it is thought that the dye contructs/polybranched-dye molecules of the present invention are internalised or taken up by cells before internal cellular mechanisms act upon the constructs causing an increase in fluorescence.
The present inventors have observed that through the use of the dye construct/poly-branched molecules described herein, that such molecules are capable of being internalised, by certain specific cells within seconds or a few minutes. However, over time many different cell types may internalise the dye construct/poly-branched molecules described herein and as such, in order to observe the desired cells which rapidly internalise the dye construct/poly-branched molecules described herein, the detection of fluorescence should be carried out within a few minutes of administering the dye construct—typically within seconds to minutes, such as 1-30 minutes, typically 1-10 minutes. In this manner, cells which may internalise such constructs over a much longer time period, such as within hours, are not detected and hence the cells which rapidly internalise the dye constructs are readily discernable, from other cells. This is also advantageous to the patient, as they are subjected to the diagnostic procedure for as short a time as possible. Moreover, if the endoscope were removed, the site of administration of the dye construct may be difficult to relocate.
The confocal endoscope or microendoscope enables real-time in vivo human and animal imaging. The instrument couples a custom built fluorescence slit-scan confocal microscope to a fibre-optic catheter. Further teaching may be found in Thiberville et al17 and WO2008020130, to which the skilled reader is directed and the contents of which are hereby incorporated by way of reference.
In a further aspect there is provided a method of visualising a particular cell or cells within a mixture of cells in vivo using confocal endomicroscopy, comprising the step of adding a dye construct or poly-branched molecule linked fluorescent dye to said mixture of cells and observing a cell or cells which have internalised the dye construct or poly-branched molecule linked fluorescent dye by observing fluorescence from said cell or cells using a confocal fluorescence endomicroscope. Conveniently the dye construct or poly-branched molecule linked fluorescent dye may be one of the constructs described herein.
Typically the cells which may be detected by way of internalising the dye construct or poly-branched molecule linked fluorescent dye are activated neutrophils. Activated neutrophils which are characterised by degranulation and protease release may be found at sites of inflammation and may therefore be detected using confocal endoscopy techniques at a variety of locations within the body of a subject such as within in the lung, within the gastrointestinal tract, within the reproductive tract or any other endoscopically accessible orifice.
In a preferred embodiment activated neutrophils are detected in the lung of a subject. Typically the subject may be a subject already hospitalised, such as a patient in intensive care, where early detection of such activated neutrophils would be desirable.
The dye construct or poly-branched molecule linked to fluorescent dye may be of the form described in, for example, WO/2003/014743 (to which the skilled reader is directed and the entire contents of which are incorporated herein, by way of reference) which comprise one or more cleavage sites which are cleavable by appropriate chemical or enzyme means. Preferred molecules comprise three or more, typically six or more branches, such that a significant increase in fluorescence may be observed following dequenching of the fluorescent moieties.
In addition to the molecules described in WO2003/014743, preferred poly-branched molecules of the present invention have the generalised structures as follows:

Where R is selected from NH2, CONH2, NH2CONH—, an amino acid, OH, amino acid —CONH2.CONH2—amino acid, alkylamino, alkoxyamino, urea, thiol, carboxylic acid, or a further fluorophore moiety which may be the same or different to the other fluorescent moieties. All such groups may be directly attached to the branch point, or may be separated from the branch point by a spacer, which may be a PEG group, an alkyl or alkenyl chain, such as a C1-C10 alkyl or alkenyl. Other linking groups are described in WO2003/014743 and reference 14.
The above generalised structures schematically represent probes which may, be suitable for use in the present invention. The use of a peptide and/or polyethylene glycol (PEG) portion is intended to improve the solubility of the dendrimer/branched molecules. When present the peptide sequence may comprise an enzyme or chemical cleavage recognition sequence or may be random in the sense of not including a recognisable enzyme or chemical cleavage recognition sequence. As an alternative to the peptide and/or PEG moiety, any suitable solubilising group known in the art may be used. Without wishing to be bound by theory, when a random peptide sequence is employed, the peptide sequence is not thought to be cleaved by an enzyme present in the cell to be detected. Thus, the increase in fluorescence observed following internalisation of the molecules of the present invention comprising random peptide sequences is not thought to be due to cleavage of the peptide moiety and release of previously quenched fluorescent moieties, in contrast to previous teachings.
When an enzyme cleavable sequence is employed, the peptide sequence may be cleaved by an enzyme which may be present outside of the cell and this may result in a low amount of fluorescence being observed. However, a far greater observable increase in fluorescence is observed upon internalisation of the molecules/probes. In this manner the separation of the dequencher moiety from the fluorescent moiety, as well as other cellular mechanisms results in a significant increase in fluorescence being observed. Thus, in an embodiment where a degree of fluorescence may be observed outside of the cell it is to be understood that a detectable increase is observed (such as greater than a factor of 1.2 as compared to any fluorescence which is observed outside of the cell) when the construct in internalised. This may in fact be an advantage, as it may allow cells to be generally identified by way of a level of fluorescence being observed outside any cells, but desired cells can more easily be identified once the constructs are internalised and an increase in fluorescence observed.
Particularly preferred molecules of the present invention are selected from one of the following structures:

(AA)nPEG is to be understood as a peptide and/or PEG moiety being present. That is one or the other or both. Indeed more than one PEG may be present. The above molecules comprise a plurality of fluorescently quenched moieties, designated *F. One such fluorescent moiety is FAM, but it is to be understood that the molecules of the present invention are not limited to the use of FAM as many other fluorescent moieties may be used, such as rhodamine, cyanine dyes and BODIPY dyes. Q* is a dark quencher moiety such as DABCYL, Methyl Red, BHQ1, BHQ2 and BHQ3.
The molecules of the present invention may comprise a peptide linkage, represented in the above structures as (AA)n, where AA means any amino acid and n may be zero or is a positive integer from 1-30 such as 1-20, or 1-15. Such peptide sequences may therefore be random sequences, or conform to known sequences contained within peptides or proteins. Sequences which are recognised by the enzyme neutrophil elastase include A-A-P-V, A-A-A-P-V-K, E-E-I-Nle-R-R. Many other peptide sequences are known to the skilled addressee and may be used in probes of the present invention, examples include G-P-K-G-L-K-G (for MMP-9), V-A-D-C-A-D-Y (for proteinase 3), A-A-P-F, or F-V-T-Gnf-S-W where Gnf=nonproteinogenic 4-guanidine-I-phenylalanine) (for cathepsin G) and D-C-V-D (for Caspase). In a further aspect, the present invention provides novel dye constructs as described above for use in visualising cells, such as activated neutrophils, in vivo. There is also provided methods of preparing such molecules as described hereinafter.
The molecules of the present invention and indeed molecules described in WO/2003/014743 are initially quenched, that is they display little or no fluorescence in terms of fluorescence which may be detected from the fluorescent moiety following appropriate excitation. However, following internalisation of the molecules by the cell or cells to be detected, a de-quenching of the molecules occurs and an increase in fluorescent signal, following excitation using light of a suitable wavelength, can be detected18.
Additionally, the present inventors have observed that certain molecules of the present invention which do not have recognisably cleavable peptide sequences are nevertheless internalised by certain cells, such as activated neutrophils and a de-quenching i.e. increase in fluorescence can be observed. Without wishing to be bound by theory, it is thought that the probes may be internalised into acidified vacuolar structures that directly effect internal quenching efficiency.
Thus, in a manner different to that described, for example, by WO2003014743, the molecules of the present invention do not necessarily have to possess recognisable enzyme cleavable peptide sequences in order to visualise cells and in particular activated neutrophils.
The present inventors are able through confocal endoscopy to visualise cells in situ in vivo. As such the term “in vivo” is to be understood to relate to cells within the living body and hence is to be distinguished from visualising cells obtained from tissue samples which have been extracted or excised from the body. The present methods may be conducted on or within any organ into which an endomicroscopic catheter may be inserted. This may be, for example, the gut including the large and small intestine; arteries and veins; the respiratory system including the lungs, the brain such as via an intracranial catheter; and the reproductive system including the womb and fallopian tubes.
In a particularly preferred embodiment, the methods of the present invention may be carried out whilst visualising cells in the lung, such as in the alveolar space.
The present inventors have observed that through the use of the dye constructs/poly-branched molecules described herein, that such molecules are capable of being internalised, by activated neutrophils. Such internalisation by activated neutrophils occurs very rapidly, within a few seconds or minutes and as such activated neutrophils may be visualised within 1-30 minutes, typically 1-10 minutes of the molecules of the present invention being administered to the subject at the site of investigation, such as within the lung. As mentioned above, the methods of detection as described herein should typically be conducted within a short period of time, following local administration of the initially quenched molecules, typically within a few minutes of administration, so that only cells, such as activated neutrophils, which internalise or take up the molecules of the present invention rapidly, are detected. Other cell types may also internalise the molecules, but over a much longer period of time. Thus, following the techniques of the present invention, it is possible to rapidly detect activated neutrophils in a mixed population of cells. In a particularly preferred embodiment, it is possible to detect activated neutrophils within the lung, such as in the alveolar space, of a subject.
Moreover, due to the sensitivity and increase in fluorescence following dequenching of the fluorescent moieties, it is possible to detect fluorescence from only microdosed amounts (typically less than 100 μg) such as less than 50 μg or even 10 μg or less of the dye construct which has been administered. This is particularly advantageous in terms of certain possible regulatory issues concerning the use of larger quantities and toxicity concerns when administering any exogenous molecule—although the molecules of the present invention may not in fact be significantly toxic in any case. It is in fact particularly surprising that such low microdose amounts of molecule when administered are capable of eliciting a signal which is detectable using microendoscopy techniques.
Thus, in a further embodiment, the present invention provides a method of visualising a particular cell or cells (such as activated neutrophils) within a mixture of cells in vivo using confocal endomicroscopy, comprising the step of adding a microdose (i.e. less than 100 μg, 50 μg, or 10 μg or less) of a dye construct or poly-branched molecule linked fluorescent dye to said mixture of cells and observing a cell or cells which have internalised the dendrimer dye molecule or poly-branched molecule linked fluorescent dye by observing fluorescence from said cell or cells using a confocal endomicroscope.
The present invention also provides use of a dendrimer dye molecule or poly-branched molecule linked fluorescent dye of the present invention in an amount of less than 100 μg, 50 μg, or 10 μg or less, for visualising cells in vivo using confocal endoscopy.
There is also provided a catheter or other suitable administration device comprising a microdose (i.e. less than 100 μg, 50 μg, or 10 μg or less) amount of a dye construct or poly-branched molecule linked fluorescent dye of the present invention, for administration to a subject, such that a cell or cells is capable of being visualised by confocal endoscopy.
One potential advantage of the present invention is that it may be carried out on subjects who are being given respiratory support in terms of being administered oxygen or air and who as such may have a face mask covering their mouth and/or nose or who are being intubated. Even in such a situation, it is possible to insert a confocal microendoscopy catheter into the lungs through the nasal passage or via the endotracheal tube. In this manner the present invention can truly be carried out at the bedside, without necessarily having to move the subject unduly. Moreover, for such subjects, the ability to detect any activated neutrophils, which are a key marker of an inflammatory response is of paramount importance and as such the present invention may find particular use in being conducted on such ill patients where moving them to another location may be undesirable and/or problematic.